San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, Milan-I, Italy.
Hum Mol Genet. 2012 Sep 1;21(17):3858-70. doi: 10.1093/hmg/dds214. Epub 2012 Jun 7.
The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca(2+) uptake capacity. This defect is neither a consequence of global alteration in cellular Ca(2+) homeostasis nor of the reduced driving force for Ca(2+) internalization within mitochondria, since cytosolic Ca(2+) transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca(2+) uptake speed in Afg3l2(-/-) cells, indicating the presence of functional Ca(2+) uptake machinery. Our results show that the defective Ca(2+) handling in Afg3l2(-/-) cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca(2+) elevations, hampering the proper Ca(2+) diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2(-/-) MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca(2+) buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca(2+) homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.
线粒体蛋白 AFG3L2 在内膜上与 paraplegin 形成同源寡聚体和异源寡聚体复合物，称为 m-AAA 蛋白酶。这些复合物负责错误折叠蛋白质的质量控制，并参与 OPA1 蛋白水解切割的调节，这对于线粒体融合是必需的。AFG3L2 的突变导致脊髓小脑共济失调 28 型和儿童复杂神经退行性综合征。在这项研究中，我们证明了 AFG3L2 在小鼠胚胎成纤维细胞（MEFs）中的缺失降低了线粒体 Ca(2+)摄取能力。这种缺陷既不是细胞内 Ca(2+)稳态全局改变的结果，也不是线粒体内部 Ca(2+)内化驱动力降低的结果，因为细胞质 Ca(2+)瞬变和线粒体膜电位不受影响。此外，在通透细胞的实验中，我们发现 Afg3l2(-/-)细胞中的线粒体 Ca(2+)摄取速度未改变，表明存在功能正常的 Ca(2+)摄取机制。我们的结果表明，Afg3l2(-/-)细胞中 Ca(2+)处理的缺陷是由于呼吸功能障碍和随后的 OPA1 处理导致线粒体网络碎片化引起的。这使得许多线粒体失去了与内质网的连接，因此没有 Ca(2+)升高，阻碍了 Ca(2+)沿线粒体网络的适当扩散。通过过表达 OPA1 使 Afg3l2(-/-) MEFs 中的线粒体碎片化恢复，挽救了受损的线粒体 Ca(2+)缓冲能力，但未能恢复呼吸作用。通过将线粒体形态和 Ca(2+)稳态联系起来，这些发现为 AFG3L2 突变引起的神经退行性变的分子机制提供了新的线索。
